NMR Characterization of Lignins Isolated from Fruit and Vegetable Insoluble Dietary Fiber

Compositional information for lignins in food is rare and concentrated on cereal grains and brans. As lignins are suspected to have important health roles in the dietary fiber complex, the confusing current information derived from nonspecific lignin determination methods needs to be augmented by diagnostic structural studies. For this study, lignin fractions were isolated from kiwi, pear, rhubarb, and, for comparison, wheat bran insoluble dietary fiber. Clean pear and kiwi lignin isolates allowed for substantive structural profiling, but it is suggested that the significance of lignin in wheat has been overestimated by reliance on nonspecific analytical methods. Volume integration of NMR contours in two-dimensional 13C−1H correlation spectra shows that pear and wheat lignins have comparable guaiacyl and syringyl contributions and that kiwi lignins are particularly guaiacyl-rich (∼94% guaiacyl) and suggest that rhubarb lignins, which could not be isolated from contaminating materials, are as syringyl-rich (∼96% syringyl) as lignins from any known natural or transgenic fiber source. Typical lignin structures, including those newly NMR-validated (glycerols, spirodienones, and dibenzodioxocins), and resinols implicated as possible mammalian lignan precursors in the gut are demonstrated via their NMR correlation spectra in the fruit and vegetable samples. A novel putative benzodioxane structure appears to be associated with the kiwi lignin. It is concluded that the fruits and vegetables examined contain authentic lignins and that the detailed structural analysis exposes limitations of currently accepted analytical methods. Keywords: Dietary fiber; plant cell wall; dioxane−water milled lignin; ball-milling; acidolysis lignin; pear; kiwi; rhubarb; wheat; NMR; HSQC; HMBC; gut fermentation; lignan

NMR Characterization of Lignins Isolated from Fruit and Vegetable Insoluble Dietary Fiber

Compositional information for lignins in food is rare and concentrated on cereal grains and brans. As lignins are suspected to have important health roles in the dietary fiber complex, the confusing current information derived from nonspecific lignin determination methods needs to be augmented by diagnostic structural studies. For this study, lignin fractions were isolated from kiwi, pear, rhubarb, and, for comparison, wheat bran insoluble dietary fiber. Clean pear and kiwi lignin isolates allowed for substantive structural profiling, but it is suggested that the significance of lignin in wheat has been overestimated by reliance on nonspecific analytical methods. Volume integration of NMR contours in two-dimensional 13C−1H correlation spectra shows that pear and wheat lignins have comparable guaiacyl and syringyl contributions and that kiwi lignins are particularly guaiacyl-rich (∼94% guaiacyl) and suggest that rhubarb lignins, which could not be isolated from contaminating materials, are as syringyl-rich (∼96% syringyl) as lignins from any known natural or transgenic fiber source. Typical lignin structures, including those newly NMR-validated (glycerols, spirodienones, and dibenzodioxocins), and resinols implicated as possible mammalian lignan precursors in the gut are demonstrated via their NMR correlation spectra in the fruit and vegetable samples. A novel putative benzodioxane structure appears to be associated with the kiwi lignin. It is concluded that the fruits and vegetables examined contain authentic lignins and that the detailed structural analysis exposes limitations of currently accepted analytical methods. Keywords: Dietary fiber; plant cell wall; dioxane−water milled lignin; ball-milling; acidolysis lignin; pear; kiwi; rhubarb; wheat; NMR; HSQC; HMBC; gut fermentation; lignan

Identifying New Lignin Bioengineering Targets: Impact of Epicatechin, Quercetin Glycoside, and Gallate Derivatives on the Lignification and Fermentation of Maize Cell Walls

Apoplastic targeting of secondary metabolites compatible with monolignol polymerization may provide new avenues for designing lignins that are less inhibitory toward fiber fermentation. To identify suitable monolignol substitutes, primary maize cell walls were artificially lignified with normal monolignols plus various epicatechin, quercetin glycoside, and gallate derivatives added as 0 or 45% by weight of the precursor mixture. The flavonoids and gallates had variable effects on peroxidase activity, but all dropped lignification pH. Epigallocatechin gallate, epicatechin gallate, epicatechin vanillate, epigallocatechin, galloylhyperin, and pentagalloylglucose formed wall-bound lignin at moderate to high concentrations, and their incorporation increased 48 h in vitro ruminal fiber fermentability by 20–33% relative to lignified controls. By contrast, ethyl gallate and corilagin severely depressed lignification and increased 48 h fermentability by about 50%. The results suggest several flavonoid and gallate derivatives are promising lignin bioengineering targets for improving the inherent fermentability of nonpretreated cell walls

Fluorescence-Tagged Monolignols: Synthesis, and Application to Studying In Vitro Lignification

Fluorescence-tagged coniferyl alcohols, coniferyl alcohol γ-coupled by ethylenediamine spacers to dimethylaminocoumarin or nitrobenzofuran fluorophores, were tested as photoprobes to study the oxidase-mediated polymerization of monolignols. The fluorescent coniferyl alcohol derivatives readily underwent peroxidase-catalyzed in vitro copolymerization with coniferyl alcohol to yield fluorescent dehydrogenation polymers, the backbone polymers of which were structurally indistinguishable from polymers formed solely from coniferyl alcohol. To illustrate the use of the photoprobes, we successfully monitored in real time the complexation of coniferyl alcohol with horseradish apoperoxidase by Förster resonance energy transfer (FRET) using the protein-tryptophan near the active site and a dimethylaminocoumarin moiety as donor and acceptor fluorophores. Furthermore, mixtures of fluorescence-tagged and normal coniferyl alcohols readily diffused into isolated maize cell walls and reacted with wall-bound peroxidases to form in muro artificial lignins that could be visualized by fluorescence microscopy. Thus we anticipate that fluorescence-tagged monolignols will be useful for in vitro and in vivo studies of cell wall lignification

MOESM7 of Sugarcane transgenics expressing MYB transcription factors show improved glucose release

Additional file 7: Table S6. Primers for ZmMYB cloning, genomic PCR and qPCR. Primers designed from GenBank accessions for PCR amplification of ZmMYB31 and ZmMYB42 genes from maize cDNA (1–4), for leaf gDNA screening from ZmMYB31 and ZmMYB42 gene cassettes (5–8) and qPCR quantification of MYB gene expression in young and maturing internodes (9–10)

MOESM5 of RNAi downregulation of three key lignin genes in sugarcane improves glucose release without reduction in sugar production

Additional file 5: Table S5. Glucose (mg/g) released via limited enzymatic hydrolysis. Glucose released per gram of bagasse measured at six time points for CCoAOMT, F5H and COMT RNAi plants. The glucose released is shown with the standard error of the mean. Samples significantly different to controls after a one-way ANOVA, p < 0.05 are shown in bold. Plants for each line are listed in ascending order of total lignin content. Avg is the mean of the lines within each construct

MOESM3 of Sugarcane transgenics expressing MYB transcription factors show improved glucose release

Additional file 3: Table S2. Normalized lignin biosynthetic genes ΔCt values for MYB-expressing plants. Values normalized against average control ΔCt value for each gene. Values represent initial expression screening of leaf tissue and post-harvest expression results from young internode tissue and maturing internode tissue. NE: Normalized expression with standard error of the mean shown. Samples significantly different to the controls after ANOVA, p = 0.05, are shown in bold. Control n = 3. Plants are listed in ascending total lignin content for each line

MOESM8 of Sugarcane transgenics expressing MYB transcription factors show improved glucose release

Additional file 8: Table S7. Primers for qPCR of lignin biosynthetic genes

Flexible Method for Conjugation of Phenolic Lignin Model Compounds to Carrier Proteins

Linking lignin model compounds to carrier proteins is required either to raise antibodies to them or to structurally screen antibodies raised against lignins or models. This paper describes a flexible method to link phenolic compounds of interest to cationic bovine serum albumin (cBSA) without interfering with their important structural features. With the guaiacylglycerol-β-guaiacyl ether dimer, for example, the linking was accomplished in 89% yield with the number of dimers per carrier protein being as high as 50; NMR experiments on a 15N- and 13C-labeled conjugation product indicated that 13 dimers were added to the native lysine residues and the remainder (∼37) to the amine moieties on the ethylenediamine linkers added to BSA; ∼32% of the available primary amine groups on cBSA were therefore conjugated to the hapten. This loading is suitable for attempting to raise new antibodies to plant lignins and for screening